A common structural motif consisting of a cystine knot and a small triple-stranded @-sheet has been defined from comparison of the 3-dimensional structures of the polypeptides w-conotoxin GVIA (Conus geogruphus), kalata BI (Oldenlundiu uffinis DC), and CMTI-I (Curcurbitu muximu). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1-4 and 2-5) and the intervening polypeptide backbone, through which the third disulfide (3-6) passes. The other component of this motif is a triple-stranded, antiparallel @-sheet containing a minimum of 10 residues, XXC2, XC,X, XXC,X (where the numbers on the half-cystine residues refer to their positions in the disulfide pattern). The presence in these polypeptides of both the cystine knot and antiparallel @-sheet suggests that both structural features are required for the stability of the motif. This structural motif is also present in other protease inhibitors and a spider toxin. It appears to be one of the smallest stable globular domains found in proteins and is commonly used in toxins and inhibitors that act by blocking the function of larger protein receptors such as ion channels or proteases.

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-Conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of -conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and -conotoxin GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs.1 H NMR studies found that PIIIA adopts two conformations in solution due to cis/ trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of -conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective -conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by -conotoxins.

The growth and metabolic actions of growth hormone (GH) are believed to be mediated through the GH receptor (GHR) by JAK2 activation. The GHR exists as a constitutive homodimer, with signal transduction by ligand-induced realignment of receptor subunits. Based on the crystal structures, we identify a conformational change in the F'G' loop of the lower cytokine module, which results from binding of hGH but not G120R hGH antagonist. Mutations disabling this conformational change cause impairment of ERK but not JAK2 and STAT5 activation by the GHR in FDC-P1 cells. This results from the use of two associated tyrosine kinases by the GHR, with JAK2 activating STAT5, and Lyn activating ERK1/2. We provide evidence that Lyn signals through phospholipase C gamma, leading to activation of Ras. Accordingly, mice with mutations in the JAK2 association motif respond to GH with activation of hepatic Src and ERK1/2, but not JAK2/STAT5. We suggest that F'G' loop movement alters the signalling choice between JAK2 and a Src family kinase by regulating TMD realignment. Our findings could explain debilitated ERK but not STAT5 signalling in some GH-resistant dwarfs and suggest pathway-specific cytokine agonists.